ROCK is a critical RhoA effector involved in regulation of multiple cellular signaling pathways through phosphorylation of multiple substrates, including myosin light chain (MLC), LIM Kinase, MYPT1, and CPI-17. ROCK substrates are involved in smooth muscle contractility, vascular permeability, actin filament organization, cell adhesion, cell migration, growth control and cytokinesis. The direct involvement of ROCK in smooth muscle contractility results from increased phosphorylation of both MLC, which increases contractility upon phosphorylation, and MLC-phosphatase, which is inhibited upon phosphorylation, thereby further increases MLC phosphorylation ROCK activation, and thus, results in increased smooth muscle contractility.
Literature reports suggest a link between ROCK activity and numerous cardiovascular diseases associated with vasotonic dysfunction, including hypertension, atherosclerosis, ischemic stroke, coronary vasospasm, cerebral vasospasm, angina, erectile dysfunction, and renal disease and also a link with other smooth muscle hyper-reactivity, such as glaucoma and asthma.
Cerebral cavernous malformation (CCM) syndrome is a disease associated with defective endothelial junctions predominantly in the brain predisposing patients to a lifetime risk of seizures, hemorrhagic stroke, and other neurological deficits. Cerebral cavernous malformations (CCMs) are vascular malformations mostly found within the central nervous system, e.g., brain stem and spinal cord, that can occur in sporadic or autosomal dominant inherited forms, the latter of which map to three loci, KRIT-1/CCM1, MGC4607/OSM/CCM2, and PDCD10/CCM3 (e.g., see Glading et al., (2007) J. Cell Biol. 179:247-254).
CCM1, CCM2 and CCM3 proteins each interact with a number of proteins, including Rap1, a RhoA-GTPase. Rap1 plays a role in regulating the actin cytoskeleton by stimulating the formation of actin fibers. Inactivating mutations in CCM1, CCM2 or CCM3 are sufficient to induce CCM vascular lesions by inhibiting endothelial cell vessel-like tube formation and extracellular matrix invasion leading to weakened junctions between cells and increased leakage from blood vessels. The loss of expression of the CCM1, -2, or -3 proteins causes a marked overexpression and increased activation of Rap1. Increased Rap1 activation is associated with increased Rho kinase-dependent (ROCK) phosphorylation, and short hairpin RNA knockdown of ROCK rescued endothelial cells from CCM pathology (e.g., see Borikova et al., (2010) J. Biol. Chem. 285:11760-11764).
CCM patients unfortunately have limited treatment options and there are no approved drugs in the United States for treating CCM. Fasudil hydrochloride, an isoquinoline sulfonamide derivative, is a potent ROCK inhibitor that has been approved outside the United States for treating cerebral vasospasm after subarachnoid hemorrhage and symptoms of cerebral ischemia. Fasudil has been used off-label by physicians to treat CCM, presumably by inhibiting ROCK kinase activity; however, Fasudil, which shows poor selectivity over other AGC-family kinases, also exhibits a number of serious toxic side effects and drawbacks including mandated short-course treatments, low oral bioavailability, cell toxicity and blood pressure fluctuations (e.g., see Xin et al., (2015) BioSci Rep. 35: 1-13).
There is a serious unmet medical need to treat patients having CCM, and more broadly for numerous other diseases involving elevated smooth muscle contractility, such as cardiovascular disease, with prevailing evidence pointing towards selective ROCK kinase inhibitors as a likely source for pharmacological therapeutic intervention. In conjunction, there is a need to develop new ROCK kinase inhibitors that have improved treatment times, improved oral bioavailability, reduced cell toxicity and reduced off-target activity, particularly over the AGC-family kinase members.